Abstract Clathrin-mediated endocytosis is the main port of entry into our cells for medically relevant substances including cholesterol-laden particles and viruses such as influenza and hepatitis. By engulfing signalingreceptors,thisfundamentalcellularprocessalsotunesoursensitivitytothepotentiallypathological actions of growth factors and neuromodulators. As such, understanding how the underlying endocytic machineryisregulatedpromisestorevealnovelmechanismsthatcouldbeharnessedtocontrolneoplastic, neurodegenerative, cardiovascular, and viral diseases. At the heart of the endocytic process lies the AP2 clathrin adaptor complex which appears to undergo a conformational change during vesicle formation to actively couple membrane and cargo to the clathrin coat. Despite the central role of AP2, we lack critical detailsabouthowthismolecularmachineisregulatedinvivoandhowthisregulationinfluencesmulticellular systems. To address this need, we have developed innovative tools in C. elegans that allow us to quantify AP2activityatmultiplelevelsandhaveemployeddeepgeneticscreenstoidentifythreeconservedprotein families that appear to govern AP2 conformation and activity. Our goal is to illuminate how these allosteric regulatorsoftheendocyticmachineryfunctionmechanistically.InAim1wewillvalidateourhypothesisthat adaptiN-Ear-BindingCoat-AssociatedProteins(NECAP)scounteracttheactive(open)conformationofAP2 to ensure proper recycling of adaptor complexes. We have discovered that AP2 accumulates in an active stateinNECAPmutants,andthatNECAPsspecificallybindopen,phosphorylatedformsofAP2.Usingcryo- EMwehavedeterminedthatthephosphorylatedAP2coreboundtoNECAPisconformationallyinactive.We willvalidatethisstructureinvivoandwhetheritreflectstheendproductofNECAPactivity.Previouslyitwas thoughtthatmembranephospholipids,cytosoliccargodomains,andphosphorylationbytheAP2-associated kinase (AAK1) activate AP2. Our preliminary data indicate that a conserved region of the membrane- associated Fer/Cip4 Homology Domain-only (FCHo) proteins is required to promote endocytosis by convertingAP2toanactivecomplex.WehavenamedthisfunctionallyimportantdomaintheAP2Activator, or APA. In Aim2 we will determine where the APA bindsAP2 using cryo-EM andtest whether the APA is sufficienttoinduceastructuralrearrangementofAP2,aswellasdefiningtherolesofmembrane,cargo,and phosphorylation in that process. We will evaluate the physiological significance of AP2 phosphorylation by characterizingkinasemutants.InournewAim3wewillexaminehowmembranetraffickinginfluencestissue physiologyusingoursuiteofassaystostudyanovelmutantinatissuepatterninginversin/nephronophthisis- 2proteincalledMLT-4thatphenocopieslossofAP2activity.Thelong-termimpactoftheproposedresearch willbetoclarifyhowfundamentalcellularmachineryiscontrolledwithspatiotemporalprecisioninmetazoans, wheremisregulationleadstoimportantdiseases.